PROJECT SUMMARY/ABSTRACT HIV/AIDS remains a debilitating disease globally, with infections among young adults in the US in recent years increasing. Although extensive efforts are dedicated to HIV vaccine and cure research, these approaches have yet to yield candidates for routine clinical use. By contrast, combination antiretroviral therapy (cART) has been used to reduce disease burden and mortality since its introduction into the clinic in the mid-1990s. Recommended cART formulations contain an integrase inhibitor that inhibits the enzyme active site and its strand transfer activity (integrase strand transfer inhibitor or INSTI). Despite their resounding success, incidence of resistance to second-generation INSTIs is increasing, and will predictably increase further as these drugs are rolled out for global usage. Paralleling the success of active site and allosteric site inhibitors of the reverse transcriptase enzyme, the clinic will benefit greatly from the addition of a second class of integrase inhibitor, such as allosteric integrase inhibitors (ALLINIs). This grant over the current funding cycle made seminal contributions to understanding the mechanism of action of pre-clinical ALLINI compounds, and such compounds are today in development at pharmaceutical companies. In this grant application we will continue to categorize the mechanism of ALLINI action, which is critical basic information required in advance of clinical rollout and clinical drug resistance. This research will in part be focused on the mechanism of action of the integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75, which helps to guide the virus to active genes for integration. In particular, some of the best-studied ALLINI chemotypes are effective inhibitors of the LEDGF/p75-integrase binding interaction. Inspired by the success of LEDGF/75 binding site ALLINI compounds, we will now characterize in detail interactions of additional host factors that are shown to bind integrase. As evidenced by the large variety of mutations that cause pleiotropic replication catastrophe, HIV-1 integrase is extremely sensitive to change. Characterization of novel host factor-integrase complexes will define new targets for future antiretroviral inhibitor development.